Basic fibroblast growth factor (bFGF) is a protein which exhibits potent mitogenic activity on a wide variety of cell types including capillary endothelial cells. The complete amino acid sequence for bFGF derived from bovine pituitary has been determined (Esch, F., et al., Proc Natl Acad Sci (USA) (1985) 82:6507). Cloned DNA sequences encoding human bFGF have been isolated and the amino acid sequences determined for 131-, 146- and 154-amino acid forms of the human protein (PCT application US86/01879, published as WO 87/01728 on Mar. 26, 1987; Abraham, J. et al., Science (1986) 233:545; Abraham, J. et al., The EMBO Journal (1986) 5:2523). Analysis of the cloned DNA sequences also demonstrated that a potential initiating methionine codon lies immediately upstream of the coding sequence for the 154-amino acid form of bFGF indicating that (i) the primary translation product from this gene is 155 residues in length, and (ii) the 154-amino acid form is derived by post-translational removal of the initiating methionine. Subsequently, Florkiewicz, R. and Sommer, A. (Proc Natl Acad Sci (USA) (1989) 86:3978-3981) and Prats, H., et al. (Proc Natl Acad Sci (USA) (1989) 86:1836-1840) reported the existence of longer forms of bFGF which may be produced as the result of alternative translation initiation at leucine codons lying upstream of the methionine initiation codon for the 155-residue primary translation product.
Due in part to its potent mitogenic activity on capillary endothelial cells, bFGF promotes angiogenesis, i.e. the process of forming new capillary blood vessels. It is, therefore, quite useful as a wound healing agent in applications where it is necessary to form a new capillary bed if the wound is to heal properly.
The availability of isolated, cloned DNA sequences encoding human bFGF has made it possible, using the techniques of recombinant DNA technology, to express the protein in host cells transformed with expression vectors containing these sequences and to recover the protein for clinical use. It has been observed that expression of the 155-residue primary translation product of human bFGF and the bovine equivalent in both prokaryotic and eukaryotic hosts capable of processing off the N-terminal methionine results in the production of protein having a microheterogeneous N-terminus (see, e.g., Barr, Philip J. et al., J Biol Chem (1988) 263 (31):16471). We have consistently observed that expression of the 155-residue primary translation product of human bFGF in E. coli results in the recovery of protein having a mixed N-terminal sequence Ala-Ala-Gly-Ser-Ile-/Ala-Gly-Ser-Ile- in approximately a 70/30 ratio. Although this microheterogeneity does not appear to affect the bioactivity of the molecule, it is generally considered desirable for clinical use to obtain a homogeneous material, i.e. a protein having essentially the same N-terminal sequence from molecule to molecule.